Following injury, tissue factor (TF)-bearing cells are exposed to the blood, triggering blood clot formation. Blood clots are stabilized by a web-like network of fibrin, which protects the clot from the rigors of flowing blood. Fibrin also has immunological functions during infection and contributes to wound healing. Studies have shown that abnormalities in the formation or quality (structure and/or stability) of the fibrin network are an established, independent risk factor for abnormal blood clots (thrombosis and/or bleeding). We and others have shown that different cells determine fibrin quality via their procoagulant activity and receptor expression. Interestingly, both cellular procoagulant activity and receptors produce fibrin that is denser and more stable near the cell than further away from the cell surface. We hypothesize that the spatial location of thrombin generation influences clot structure and stability. We will use laser scanning confocal microscopy to characterize activities that regulate fibrin clot structure proximal and distal to the site of clotting initiation. We will specifically investigate the roles of cell stimulation, cellular and circulating tissue factor, soluble clotting factors, and cell receptors in determining clot structure. We will use passive and magnetically-driven microbead techniques and microscopy to examine how vascular cells modulate fibrin network permeability, mechanical strength and resistance to fibrinolysis. Finally, we will use transmission electron microscopy to compare fibrin structure in clots formed under different shear rates on freshly-excised human vessels. We anticipate our data will show how different hemostatic and prothrombotic mechanisms contribute to clot structure and stability in static and low shear conditions. These mechanisms may be specific targets for preventing intravascular thrombosis and/or embolism. PUBLIC HEALTH RELEVANCE: Inappropriate blood clot formation (thrombosis) is the leading cause of death and disability in the Western world. Understanding the mechanisms leading to thrombosis is critical in order to develop approaches for preventing this devastating disease.